The present invention relates to recombinant vectors, recombinant viruses, transformants, canine interleukin 18, canine interleukin 1xcex2 converting enzyme and interleukin 18 production methods, where the objective is to mass produce by genetic manipulation.techniques canine interleukin 18 the protein primary structure of which is derived from canine genetic information and to thus make pharmaceuticals for animal use (antitumour drugs/anti-allergy drugs/antiviral drugs/vaccine adjuvants).
Interleukin 12 (hereinafter abbreviated to IL12), which shows immune control action, is a cytokine which possesses actions comprising interferon xcex3 inducing activity and physiological activity in regard to, for example, activating natural killer cells and type 1 helper T cells (reference 1) and, in particular, as a result of a powerful activating action in respect of cellular immunity, it is regarded as having very good prospects as an antitumour drug and anti-allergy drug candidate (references 2 and 3). Interleukin 18 (hereinafter abbreviated to IL18) has also been recently cloned as a cytokine showing the same kind of activity as IL12 (reference 4), and it has been reported that due to synergistic action with IL12 the activities thereof are further increased (reference 5).
Following mouse IL18 (reference 4), human IL18 cDNA has also been cloned (reference 6) by genetic manipulation techniques, and mass production using these gene recombination techniques has been investigated. However, IL18 does not have a signal sequence required for secretion from within cells. Hence, in order to be secreted from within cells in the active form, processing of an IL18 precursor protein by means of an interleukin 1xcex2 converting enzyme (hereinafter abbreviated to ICE) is necessary and so, when the IL18 gene is introduced on its own into animal cells, it is not expressed as the active form of IL18 (reference 7) and therefore the efficient mass production of recombinant form IL18 using cells has been difficult.
Development as remedies for tumours, allergies and viral diseases, etc, may be expected by means of mass production using IL18 gene recombination techniques.
In pets, particularly dogs, in common with humans, there are known many tumours such as mammary gland tumours, allergic dermatitis, and numerous viral diseases such as parvovirus infection and distemper infection, and the development of remedies for these is desired.
The cloning of canine IL18 has not yet been reported. Hence, if canine IL18 could be cloned, it is possible that it could form a novel canine remedy.
Again, if it were possible to readily mass produce IL18 in cells using gene recombination techniques, then there could be expected to emerge L18 applications as, for example, human and animal antitumour drugs, anti-allergy drugs and antiviral drugs, etc.
Against this background, with the objective of cloning canine IL18 cDNA and large-scale expression of the IL18 gene, and based on an original concept, the present inventors have succeeded in cloning the gene coding for canine IL18 from canine cDNA, and furthermore they have produced a recombinant baculovirus containing DNA coding for an IL18 precursor protein, and it has been discovered that by infecting insect cells or larvae with this, surprisingly, the active form of IL18 is produced without ICE treatment, and it has been further discovered that by producing a gene in which there is added the gene coding for the signal sequence in front of the gene coding for the active form of canine IL18, the level of production of the active form of IL18 is further enhanced. Furthermore, the gene coding for canine ICE has been successfully cloned from canine cDNA and a recombinant baculovirus containing at the same time DNA coding for a canine IL18 precursor protein and DNA coding for canine ICE has been produced, and canine IL18 has been successfully mass produced by infecting insect cells or larvae, and hence a method for producing IL18 simply and on a large scale has been established. The present invention has been perfected based on these discoveries.
Specifically, the present invention relates to canine interleukin 18 having at least one of the abilities selected from an ability to act on canine leukocytes and induce antiviral active factors and factors which enhance class II MHC expression on canine tumour cells; an ability to promote the proliferation of canine lymphocytes; an ability to enhance Fas ligand expression on canine lymphocytes and canine tumour cells; an ability to obstruct and destroy canine tumour cells; an ability to bring about a reduction in size of tumours occurring in the bodies of dogs; and an ability to activate canine leukocytes and suppress canine allergies.
Furthermore, the present invention offers recombinant vectors which bring about the production of IL18, Escherichia coli transformants possessing these recombinant vectors, recombinant baculoviruses which bring about the production of IL18 in insect cells or larvae, IL18 obtained therefrom, and also an IL18 production method. Moreover, the present invention also offers a gene coding for canine IL18, canine ICE and a gene coding for canine ICE. Furthermore, it offers a canine immune disease remedy containing IL18.
A recombinant vector in which there has been inserted DNA coding for canine IL18 of the present invention can be produced, for example, as follows. After extracting poly (A) RNA from canine cells, cDNA synthesis is carried out and, using primers based on the gene sequences coding for mouse or human IL18, polymerase chain reactions (hereinafter abbreviated to PCR) are conducted.
Furthermore, from the synthesized cDNA a phage library is produced and, by carrying out plaque hybridization with the gene fragments obtained by PCR, full length canine IL18 cDNA can be cloned. Full length canine ICE cDNA can be cloned similarly.
As methods for obtaining RNA from canine organs and the like, there are the usual methods such as, for example, those employing polysome isolation, sucrose density gradient centrifugation and electrophoresis. The extraction of the RNA from the aforesaid canine organs and canine cells can be carried out by selection of a suitable method from amongst the guanidine thiocyanate-cesium chloride method where guanidine thiocyanate treatment is carried out followed by CsCl density gradient centrifugation (reference 8), the method of phenol extraction following treatment with a surfactant in the presence of a ribonuclease inhibitor, using a vanadium complex (reference 9), the guanidine thiocyanate-hot phenol method, the guanidine thiocyanate-guanidine hydrochloride method, the guanidine thiocyanate-phenol chloroform method, and the method where treatment with guanidine thiocyanate is carried out followed by treatment with lithium chloride, and RNA precipitation effected.
From canine organs and, for example, mitogen-stimulated canine monocytes and lymphocytes, mRNA is isolated by the usual methods, for example the lithium chloride/urea method, the guanidine isothiocyanate method and the oligo dT cellulose column method, etc, and cDNA is synthesized from the mRNA obtained by the usual methods, for example by the method of Gubler et al. (reference 10) or the method of H. Okayama et al. (reference 11). To synthesize cDNA from the mRNA obtained, basically besides using avian myeloblastosis viral (AMV) or other such reverse transcriptase, there may be combined methods where DNA polymerase or the like is employed using partial primers. The use of commercial synthesis or cloning kits is convenient.
Using this cDNA as a template, PCR is carried out using primers based on mouse or human base sequences and furthermore, after ligating the synthesized cDNA to a xcex3 phage vector, packaging is carried out by mixing in vitro with xcex3 phage coat protein, etc, and the E. coli which constitutes the host is infected with these created phage particles. In such circumstances, xcex3 phage-infected E. coli undergoes lysis and individual clones are recovered as plaques. These plaques are transferred to nitrocellulose or other such filters and, by hybridization using as probes radioactively labelled genes obtained by PCR, the canine IL18 and canine ICE can be cloned.
Procaryotes or eucaryotes can be used as the hosts. As procaryotes, there can be used bacteria, in particular Escherichia coli and bacteria of the genus Bacillus, for example Bacillus subtilis, etc. As eucaryotes, there can be used eucaryotic microorganisms such as yeasts, for example yeasts of the genus Saccharomyces, e.g. Saccharomyces serevisiae or the like, insect cells, for example Spodoptera frugiperda cells, Trichoplusiani cells and Bombyx mori cells, and animal cells, for example human cells, monkey cells, mouse cells and the like. In the present invention, there can furthermore be used the organisms themselves, for example insects such as Bombyx mori, Trichoplusiani and the like.
As expression vectors, there can be used plasmids, phages, phagemids and viruses (baculo (insect), vaccinia (animal cells), etc). Promoters within the expression vectors are selected depending on the host, for example as promoters for bacteria, the lac promoter, trp promoter and the like, can be used and, as promoters for yeasts, for example the adh1 promoter, pak promoter and the like, can be used. Furthermore, as promoters for insects, there are the baculovirus polyhedrin promoter and the p10 promoter, and for animal cells there are the simian virus 40 early or late promoter, and the like, but there is no restriction thereto.
Host transformation by means of the expression vectors can be carried out by conventional procedures well known to those skilled in the art. These methods are described in, for example, Current Protocols in Molecular Biology, published by John Wiley and Sons. Culture of the transformants can also be carried out in accordance with conventional procedure.
When IL18 is produced using cells, because the signal sequence is not present in the precursor protein, IL18 precursor protein processing by means of ICE or the like, is necessary. Consequently, by simultaneously bringing about expression in terms of the IL18 precursor protein and ICE, the active form of IL18 can be produced. For example, by using an expression vector simultaneously containing the gene coding for canine IL18 and the gene coding for canine ICE shown in Sequence No: 2, the active form of canine IL18 can be produced in cells.
Furthermore, by adding a gene coding for the signal sequence in front of the gene coding for the active form of IL18, the active form of IL18 can be produced. For example, by using a gene containing the gene coding for the signal sequence as shown in Sequence No: 9, the active form of canine IL18 can be produced in cells.
Canine IL18 can be produced using a Bombyx mori expression system, for example by producing recombinant Bombyx mori nuclear polyhedrosis virus which infects Bombyx mori. The recombinant Bombyx mori nuclear polyhedrosis virus can be produced by cotransfecting a Bombyx mori established cell line with a recombinant plasmid produced by ligating DNA coding for the canine IL18 protein in a Bombyx mori cloning vector and Bombyx mori nuclear polyhedrosis viral DNA. Hence, the recombinant virus can be produced by an in vivo method.
Specifically, there can be produced a recombinant plasmid in accordance with normal genetic manipulation where the DNA portion coding for the canine IL18 protein is ligated downstream of the expression control portion of a Bombyx mori cloning vector such as, for example, pBK283. After cotransfecting a Bombyx mori established cell line, for example BM-N strain (reference 12), by methods such as those in the literature with this recombinant plasmid and Bombyx mori nuclear polyhedrosis viral DNA (reference 12), culture is continued and the recombinant virus can be cloned from amongst the non-recombinant (wild type) and recombinant viruses appearing in the culture fluid by the usual methods such as the limiting dilution method or the plaque method. Since the recombinant virus does not have a polyhedrin forming capability, it can be readily distinguished from the wild type virus. The production of canine IL18 is carried out by bringing about the proliferation of the aforementioned recombinant Bombyx mori nuclear polyhedrosis virus within a Bombyx mori established cell line or within Bombyx mori organisms.
When a Bombyx mori established cell line is used, BM-N cells are infected using culture fluid containing the aforementioned-recombinant virus and cultured by attached culture or suspension culture. For the culture medium used to culture the BM-N cells there can be used, for example, TC-10 medium to which bovine serum has been added (reference 12). Regarding the culture temperature, 25-28xc2x0 C. is suitable. After culture, the culture fluid is centrifuged and the canine IL18 is recovered from the supernatant.
When Bombyx mori organisms are used, culture fluid containing the aforementioned recombinant virus is injected into Bombyx mori larvae, synthetic feed is given and rearing carried out. After rearing, the body fluid is collected and canine IL18 is recovered from the supernatant thereof.
When determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, the apparent molecular weight of the canine IL18 protein produced is approximately 15-20 kD.
As shown in the following examples, the canine IL18 is characterized by activities such as a capacity for inducing canine IFN xcex3 from canine leukocytes, an expression enhancing capacity in respect of Fas ligand molecules on tumour cells and an antitumour action in respect of canine tumour cells. Canine IFN xcex3 activity is measured on the basis of antiviral activity using the CPE method (reference 14) and expression enhancing activity in respect of class II MHC molecules on canine tumour cells. Regarding the expression enhancing activity in respect of Fas ligand molecules and the expression enhancing activity in respect of class II MHC molecules on tumour cells, fluorescence labelled antibodies to these molecules are reacted with the cells and the intensity of fluorescence is measured by means of a device for measuring fluorescence intensity such as a flow cytometer, and activity can be judged to exist if this increases by 10% or more.